PROJECT SUMMARY/ABSTRACT Mitochondria are essential regulators of energy metabolism, signaling pathways, and cellular homeostasis and have been implicated in the pathology of multiple age related diseases. The long-term goal of our research is to elucidate systems that regulate mitochondrial gene expression and function. A full understanding of mitochondrial regulatory factors, and how this system is altered with age and disease states, may inform therapeutic interventions. microRNAs (miRNAs) are a class of evolutionarily conserved small non-coding RNAs that are involved in post-transcriptional gene regulation. In humans, miRNAs are loaded into one of four Argonaute (AGO) proteins and target mRNAs for silencing. Differential expression of miRNAs provide a system for cells to maintain homeostasis or adapt to altered cellular conditions. Recently, miRNAs and AGO2 have been discovered inside mitochondria, presenting a potentially novel mode of mitochondrial DNA (mtDNA)-encoded gene regulation that may be used to modulate oxidative phosphorylation (OXPHOS). Of the four AGO proteins found in humans only AGO2 is catalytically active and capable of endonucleolytic cleavage of mRNA targets; interestingly this is the only AGO protein identified in the mitochondria. AGO2 has been reported to interact directly with mitochondrial ribosomal rRNAs (Zhang et al. 2014; Jagannathan et al. 2015) and associate with additional mitochondrial proteins involved in mitochondrial ribosome assembly and translation. The objective of this proposal is to determine how mitochondrial-localized miRNAs (mitomiRs) and AGO2 are involved in the regulation of mtDNA expression, and establish whether cellular conditions relevant to aging alter the mitochondrial-specific function of these factors. First I will develop a system in which I can strictly control the localization of AGO2 in order to probe its mitochondrial-specific function. Next I will determine the miRNA-dependent and independent functions of AGO2 in specific steps of mitochondrial gene expression. Using this system I will test the effects of calorie restriction in regulating the function of mitomiRs and mitochondrial-localized AGO2 in mice. CLIP-Seq will be used to identify new mitomiRs recruited under these conditions. In order to understand the biological effects of novel mitomiRs I will identify their direct mtDNA-encoded targets and determine how regulation modulates mitochondrial function. Completion of this project will provide insight into a novel mode of mitomiR based mtDNA regulation. By studying this system under conditions that are relevant to aging, it is expected that new regulators of mitochondrial function that play a role in age related diseases will be identified, which could lead to the development of novel biomarkers and therapeutic targets for aging.